Correction: Zebrafish atoh8 mutants do not recapitulate morpholino phenotypes.

[This corrects the article DOI: 10.1371/journal.pone.0171143.]

Zebrafish <i>atoh8</i> mutants do not recapitulate morpholino phenotypes

<div><p>Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it has been implicated in the regulation of pancreatic and endothelial cell differentiation, the phenotypic consequences of Atoh8 loss are uncertain. Conclusions from knockout studies in the mouse differ widely depending on the targeting strategy used, while <i>atoh8</i> knockdown by interfering morpholino oligonucleotides (morpholinos) in zebrafish has led to a range of developmental defects. This study characterised zebrafish embryos homozygous for <i>atoh8</i>^{<i>sa1465</i>}, a loss-of-function allele of <i>atoh8</i>, in order to provide genetic evidence for the developmental role of Atoh8 in this species. Embryos homozygous for <i>atoh8</i>^{<i>sa1465</i>} present normal body morphology, swimbladder inflation, and heart looping, and survive to adulthood. These embryos do not develop pericardial oedema by 72 hpf and are not sensitised to the loss of Fog1 protein, suggesting that this previously described abnormality is not a specific phenotype. Vascular patterning and primitive haematopoiesis are unaffected in <i>atoh8</i>^{<i>sa1465/sa1465</i>} mutant embryos. Together, the data suggest that Atoh8 is dispensible for zebrafish development under standard laboratory conditions.</p></div

<i>sa1465</i> embryos have normal vascular patterning and caudal vein plexus formation.

<p>a) Confocal z-stacks of WT and sa1465 embryos at the indicated stages. Insets (top) = caudal vein plexus region of 29 hpf embryos. b) qPCR for vascular markers in <i>WT</i> and <i>sa1465</i> embryos at 29 and 72 hpf.</p

Vascular expression of <i>atoh8</i>, and regulation by Bmps.

<p>a) <i>atoh8</i> expression in 24 hpf Tg(<i>kdrl</i>:eGFP) embryos. b) <i>atoh8</i> expression in the blood island of 29 hpf embryos. c) Levels of <i>atoh8</i> staining in caudal vein plexus (CVP) region of 29 hpf zebrafish embryos. Tg(<i>kdrl</i>:eGFP) embryos treated with DMSO, 5 μM LDN-193189, or 10 μM DMH4. Number of embryos: 50 (DMSO), 66 (LDN-193189), 30 (DMH4).</p

No alteration in response to atoh8 or fog1 knockdown in atoh8^{sa1465/sa1465} mutants.

<p>a) Atoh8 Western blot on products from <i>in vitro</i> transcription/translation reactions (TNT Quick) seeded with atoh8 morpholino at the indicated concentrations. The full length membrane can be viewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171143#pone.0171143.s002" target="_blank">S2 Fig</a>. b) Percentage of <i>WT</i> and <i>sa1465</i> embryos displaying pericardial oedema at 72 hpf, following injection with the indicated morpholinos. c) Examples of pericardial oedema in <i>WT</i> and <i>sa1465</i> embryos injected with atoh8, id3, and fog1 morpholinos. Imaged at 72 hpf.</p

Atoh8^{sa1465/sa1465} mutants are morphologically normal, with correct heart looping.

<p>a) Structure of the zebrafish <i>atoh8</i> locus and Atoh8 protein. The positions of the A > T substitution in the atoh8^sa1465 allele, and of Lysine 100 in Atoh8 protein, are indicated with asterisks. The red arrowheads mark the positions of each Methionine in the Atoh8 protein, and the basic (orange) and HLH (yellow) domains are indicated. The truncated protein Atoh8^K100X is the predicted product of atoh8^sa1465. Human ATOH8 contains additional proline-rich (blue) and serine-rich (green) domains. b) Atoh8 Western blot on products from <i>in vitro</i> transcription/translation reactions (TNT Quick) using atoh8^WT/WT and atoh8^{sa1465/sa1465} as templates. Predicted size of Atoh8 = 29.8 kDa. Note that there is a strong nonspecific band at ~31 kDa. The full length membrane can be viewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171143#pone.0171143.s001" target="_blank">S1 Fig</a>. c) qPCR on atoh8^WT/WT and atoh8^{sa1465/sa1465} embryos (hereafter labelled as '<i>WT</i>' and '<i>sa1465</i>', respectively). * <i>p</i> = 0.028. d) <i>WT</i> and <i>sa1465</i> embryos at 5 days postfertilisation, showing normal overall body morphology and swimbladder inflation. e) Confocal z-stacks showing <i>WT</i> and <i>sa1465</i> embryo heart morphology.</p

Normal primitive haematopoiesis in <i>sa1465</i> embryos.

<p>a) O-dianisidine staining in 48 hpf <i>WT</i> and <i>sa1465</i> embryos. b) Number of peroxidase-positive cells present in the distal tail (caudal to the cloaca), in 48 hpf <i>WT</i> and <i>sa1465</i> embryos. c) Examples of peroxidase staining in 48 hpf <i>WT</i> and <i>sa1465</i> embryos. d) qPCR for blood markers in <i>WT</i> and <i>sa1465</i> embryos.</p

Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes.

Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos

Mutation of the zebrafish <i>tmem88a</i> gene.

<p>(A) Schematic structure of the zebrafish <i>tmem88a</i> gene shows three exons and two introns. TALEN pairs TAL1 (green) and TAL2 (magenta) were designed and assembled to target the coding region of exon2. There is a ScaI endonuclease restriction site within the spacer region (orange). (B) A <i>tmem88a</i> mutant line with an 8 bp deletion (Δ8) was generated. (C) Predicted translated sequences of wild-type Tmem88a and Tmem88aΔ8 show frame-shift after residue 48 (red), causing truncation of the protein and loss of the trans-membrane domains (blue) and the Dishevelled-binding domain (teal). (D) Brightfield images of control <i>Tg(fli1a</i>:<i>egfp)</i> and <i>tmem88a</i>Δ8 embryos at 24 hpf (n = 67, n = 53 respectively), 2 dpf (n = 54, n = 35), and 5 dpf (n = 51, n = 18) as labelled. (E) Sequence alignment shows that the <i>tmem88a</i> e2i2 SBMO is specific to <i>tmem88a</i> and illustrates a 9 bp mismatch with <i>tmem88b</i> mRNA.</p

Erythropoiesis was not affected in <i>tmem88a</i>Δ8 zebrafish mutants.

<p>(<b>A-D</b>) o-Dianisidine staining of erythrocytes at 48 hpf in uninjected <i>Tg(fli1a</i>:<i>egfp)</i> controls (A, n = 16) or controls injected with <i>tmem88a</i> SBMO (B, n = 12), and uninjected <i>tmem88a</i>Δ8 mutants (C, n = 18) or mutants injected with <i>tmem88a</i> SBMO (D, n = 15). (<b>E</b>) The area of o-Dianisine staining was quantified and presented as a percentage of the total yolk area, shown in a Tukey box and whisker plot. No significant difference was found between control embryos and <i>tmem88a</i>Δ8 mutants (<i>p</i> = 0.2710, Unpaired Student’s t test). (<b>F</b>) <i>tmem88b</i> expression is not affected in <i>tmem88a</i>Δ8 mutants. qRT-PCR showing the expression of <i>tmem88b</i> in <i>tmem88</i>Δ8 mutants compared to <i>Tg(fli1a</i>:<i>egfp)</i> controls at 13 and 48 hpf. <i>P</i>-value determined by Unpaired Student’s t test. Error bars denote the standard deviation of three biological replicates. A, anterior; h, hours post fertilisation; D, dorsal; L, lateral; V, ventral; n.s., not significant.</p